Introduction

Molecular imaging emerges as an intriguing technology which plays increasingly significant roles in early diagnosis and therapeutics (Hellebust and Richards-Kortum 2012). With the help of this promising tool, clinicians worldwide are able to visualize disease progress at cellular, even molecular levels. Molecular imaging, in comparison with traditional diagnostic imaging methods, tries to spot the molecular abnormalities via probes rather than obtain the images of the end effects of molecular variation. Molecular probe is an agent frequently used to unveil the biological processes that make them visible both in vitro and in vivo in the living subjects (Reynolds and Kelly 2011). Typically, a probe is composed of three parts: signal agent, target agent, and linker (Ye et al. 2011a). At present, nanotechnology has become an active approach in many fields, and the emergence of new-type nanoparticle leads to the transformation of molecular imaging (Minchin and Martin 2010; Bai 2013) due to its unique optical and magnetic properties (He et al. 2008; Rosenblum et al. 2010). Nanoparticle usually is an ultra-fine particle with diameter varying from 1 to 100 nm, and can be distinguished from other bulk material owing to its unique properties (Mohanpuria et al. 2008). Various types of nanoparticle, including upconversion nanoparticles (UCNPs), quantum dots (QDs), dye-doped nanoparticles, and magnetic nanoparticles, hold promise as biomedical imaging, diagnostic, and theragnostic agents (Alivisatos 1996; Choi and Frangioni 2010; Kalayci et al. 2010, 2013; Hazer et al. 2011, 2012; Hazer and Hazer 2011; Liu et al. 2012; Zeng and Miao 2009). The typical near-infrared (NIR) fluorescent properties of UCNPs, QDs, and dye-doped nanoparticle make them a potential NIR probe. In contrast with visible light excitation, NIR light excitation for in vivo imaging has several desirable advantages, namely weak autofluorescence, deep penetration, minimal photo-bleaching, etc. In magnetic resonance imaging (MRI), superparamagnetic iron oxide nanoparticle (SPIO) is one of most commonly used signal agent. MRI is a powerful diagnostic imaging modality with high space resolution. It can visualize the tissue structures by employing the magnetic resonance of protons. SPIO, which becomes magnetic in an external magnetic field but nonmagnetic when the magnetic field is removed, has been the most T2 probe for MRI (Lodhia et al. 2010; Jeon et al. 2013; Xie et al. 2011).

Even though nanoparticle has its unique advantages, some undesirable drawbacks still prohibit it being an ideal probe design platform (Ye et al. 2011b; Dutta et al. 2010). Nanoparticle can easily aggregate because of its high surface area, and for this regard, surface modification becomes necessary for keeping its stability. Practically speaking, the synthesized nanoparticle should be directly used in molecular imaging. But as synthesized, most nanoparticles are hydrophobic which is not suitable for clinical use. For example, nanoparticles modified with hydrophobic organic ligands (such as oleic acid) cannot be directly applied because of their poor hydrophilicity and lack of functional groups. With this regard, it is crucial to chemically modify the surface of nanoparticles as to make them hydrophilic and biocompatible (Sapsford et al. 2013). Anyway, surface modification is one of the most critical steps in the development of functionalized nanomaterials for bioimaging applications (Wang et al. 2010). Past decades have seen the rise of great interest in nanoparticles not only owing to their unique application in optical or MRI, but also their capability as ideal building blocks for multimodal bioimaging and therapy (Fig. 1) (Cai and Chen 2007, 2008; Gu et al. 2011; Licha and Resch-Genger 2011; Swierczewska et al. 2011; Zhou et al. 2012; Ladj et al. 2013). In this review, different methods for the preparation of different kinds of nanoparticles are summarized. We detailed on the synthesis and surface modification of nanoparticles in molecular imaging probe design, with a focus on two widely used nanoparticles (UCNPs and MNPs). Additionally, the perspectives and challenges of nanoparticle-based probe in molecular imaging are discussed.

Fig. 1
figure 1

1 In vivo upconversion luminescence imaging of subcutaneous HeLa tumor-bearing athymic nude mice (right hind leg, pointed by white arrows) after intravenous injection of UCNP-NH2 (A) and UCNPs-FA (B) (Xiong et al. 2009a). 2 (a) Scheme of a multimodal imaging probe. (b) UCL, (c, d) MRI and (e) PET (Liu et al. 2011a)

Full size image

Structure of nanoparticle-based probe

To be usual, three parts compose the molecular imaging probes: signal agent, target agent, and linker (Fig. 2) (Ye et al. 2011; Reynolds and Kelly 2011). Generally, the spectrum of target agent can range from small molecules to macro molecules, such as peptides, proteins, antibodies, etc. The signal agent encompass radionuclides (for PET, SPECT), bioluminescence or fluorescent molecules (for optical imaging), magnetic molecules (for MRI) (Lee et al. 2012; Misri et al. 2012). And a linker is employed to bridge two agents mentioned above. Different types of linker are very important in molecular imaging (Cheng et al. 2005).

Fig. 2
figure 2

Schematic illustration of general molecular probe

Full size image

Signal agent

The best representative examples in molecular imaging are MNPs and UCNPs. MNPs are the most commonly used contrast agent in MRI on account of their positive/negative enhancement effect on T1/T2 sequences. UCNPs are a kind of nanoparticle which has quite distinctive upconversion properties. Upconversion refers to the processes in which two or more low-energy input photons converted to a higher-energy output photon. When excited continuously at 980 nm under laser, UCNPs presents unique UCL (upconversion luminescence) properties, such as sharp emission lines, long lifetimes, a large anti-stokes shift, etc.

Target agent

An ideal target agent should have specific high binding affinity and selectivity with a target receptor. Importantly, the binding affinity and selectivity should be retained or not be dramatically changed after conjugation with signal agent. And even more importantly, the binding affinity or selectivity can be improved by some different ways such as dimerization, multimerization etc.

Linker

As an essential part connecting signal agent and target agent, the structural type, length, hydrophilicity of a linker can frequently influence the receptor target conjugation. What’s more, the multivalent effect of a linker in further conjugation should also be considered. In recent years, nanoparticle has been a hot signal platform due to its unique properties in probe design. Under such circumstances, linker has gotten great importance.

Synthesis of nanoparticle-based probe

Preparation of nanoparticle

Generally speaking, suitable size and good imaging performance of the nanoparticles are the primary precondition for molecular imaging (Fig. 3). To date, there are mainly three synthesis methods reported for the preparation of nanoparticles with proper size and high-quality imaging performance: the co-precipitation, hydro (solvo) thermal, and thermal decomposition.

Fig. 3
figure 3

Schematic illustration of nanoparticle (TEM)

Full size image

Coprecipitation is a relatively easy and convenient method, which is based on the chemical reactions carried out in an aqueous medium. Taking the synthesis of MNPs as example, magnetic nanoparticles can be coprecipitated by adding a base into a solution of Fe(III) and Fe(II) salts. Furthermore, the stable MNPs can be synthesized in the presence of a proper coating agent. It doesn’t need costly equipments, complex procedures, and harsh reactions (Mai et al. 2006). But it still has its own disadvantages, for example, it is difficult to obtain nanoparticles with uniform size using this method.

Hydro (solvo) thermal method is a typical solution-based nanoparticle synthesis method, which exploits a high temperature and pressure situation to increase the solubility and reactivity of inorganic particles. The advantages of this method include easy control of the reaction conditions, the relatively low cost, and high yield. By using this method, nanoparticles with uniform size can be prepared at mild temperatures. The shapes and sizes of nanopaticles can be well controlled by selecting different surfactants. In 2005, Wang introduced a general strategy for nanocrystal synthesis (Wang et al. 2005a, b). In a typical synthesis, the cocktail of noble metal salts solution, solvent, and surfactants were added to an autoclave tube under agitation. Then the reactions underwent for several hours at different temperature. When the autoclave was cooled down to room temperature, the products were collected at the bottom of the vessel. Based on the same synthesis process, the size of nanoparticle can be controlled between 10 and 100 nm.

Thermal decomposition is another common method to synthesize nanoparticles (Yi and Chow 2006; Boyer et al. 2007). For example, Fe3+ acetylacetonate or Ln3+ trifluoroacetates are decomposed in the presence of surfactants to prevent particles from aggregation (Boyer et al. 2007). Usually, the size of nanoparticle can also be well controlled using this method. However, the relatively harsh reaction conditions may be a major limit for its applications.

Surface modification of nanoparticle

Nanoparticle is typically prepared in the protection of coating materials to avoid aggregation. The surface coating of the nanoparticle can be achieved by using appropriate polymer or surfactants, e.g., poly (ethylene glycol) (PEG), dextran, carboxydextran, poly (vinyl alcohol) (PVA), polyethylenimine (PEI), oleic acid (OA), lauric acid etc.; or silane, precious metal, carbon, etc. (Butterworth et al. 2001; Sahoo et al. 2001; Berry et al. 2003; Chastellain et al. 2004; Sun et al. 2004; Lu et al. 2005; Briley-Saebo et al. 2006; Jeong et al. 2010; Liu et al. 2008, 2012; Chen et al. 2008; Cormode et al. 2009; ). Surface coating materials such as polymers or surfactants are usually added during the formation of new surfaces to prevent nanoparticle from aggregation (Durán et al. 2008). Notably, the type of the surface coating materials not only determines the size of nanoparticle but also plays an important role in further biological imaging applications (Lu et al. 2007; Reddy et al. 2012).

Among different kinds of polymer coating materials (Fig. 4), PEG is the most widely used polymer for nanoparticle coating (Zhang and Ferrari 1998; Gref et al. 2000; Shang et al. 2006.) due to its impressive properties. In order to attach PEG to nanoparticle, various methods have been established, represented by nanoparticle polymerization (Lutz et al. 2006; Flesch et al. 2005), and the surface silane grafting (Butterworth et al. 2001; Zhang et al. 2002, 2004; Kohler et al. 2004; Veiseh et al. 2005; Larsen et al. 2009; Pilloni et al. 2010). Apart from PEG, the dextran was also been extensively exploited in nanoparticle surface coating for molecular imaging (Weissleder et al. 1995; Josephson et al. 1999; Corot et al. 2006). In order to lubricate further surface conjugation between target agent and nanoparticle, the dextran coat can be cross-amino-linker with the help of epichlohydrin and ammonia (Josephson et al. 1999; Schellenberger et al. 2002; Wunderbaldinger et al. 2002).

Fig. 4
figure 4

Schematic illustration of polymer coating materials: PEG (a), PVA (b), PEI (c), chitosan (d), dextran (e)

Full size image

When taking into account of nanoparticle safety, biocompatibility, stability, and hydrophilicity, silane such as tetraethoxysilane is a good choice in nanoparticle coating (Lu et al. 2002; Tan et al. 2004; Tada et al. 2007; Bi et al. 2008; Sun et al. 2008). Precious metal (such as gold) could be used as surface coating material owing to its low reactivity. Moreover, gold surface can be modified with thiol groups for further conjugation (Colvin et al. 1992). However, disappointedly, such nanoparticle used in bioimaging should be hydrophilic, and some surface coating materials are instinctively hydrophobic (oleic acid). Therefore, much work still needs to be done in further modification on account of subsequent biological application.

A common approach to modify surface-coated nanoparticle is ligand engineering. It involves a ligand exchange reaction with hydrophilic materials or a direct oxidation of the terminal group. As an example, hydrophobic ligand-coated nanoparticle can be transformed into hydrophilic through ligand exchange reactions by a wide variety of hydrophilic agents such as 6-aminohexanoic acid (Meiser et al. 2004), hexanedioic acid (Zhang et al. 2007, 2009), citrate (Liu et al. 2010), polyacrylic acid (Naccache et al. 2009), and phosphate-derived molecules (Traina and Schwartz 2007). Apart from ligand engineering, ligand attraction, electrostatic layer-by-layer assembly, and surface polymerization can also fulfill the surface modification. Ligand attraction means attaching an amphiphilic ligand onto nanoparticle surface through the hydrophobic–hydrophobic interaction (Pedroni et al. 2011). Layer-by-layer assembly involves electrostatic attraction of oppositely charged ligand on the surface of nanoparticle (Wang et al. 2005a). Surface polymerization means growing a dense shell on the surface of nanoparticle with the help of auto-polymerize ligand (Sivakumar et al. 2006). A proper active functional group can provide easy access to nanoparticle for subsequent biological conjugation. Nevertheless, most nanoparticles lack functional groups after the hydrophilic modification. Therefore, an additional surface modification step to increase active functional group is required. The feasible solution here is connecting with a linker. Till now, many linkers have been designed and applied in the nanoparticle-based probe, and various linkers will be introduced as follows. Here, we divided linkers into several categories on the basis of their terminal functional group, such as carboxylic acid, amine, maleimide groups etc. (Zhou et al. 2012; Sapsford et al. 2013).

Carboxylic acid (–COOH) groups linker used in nanoparticle-based probe

Carboxylic acid group (–COOH) is especially useful for coupling with target agent like antibodies, folic acid, and DNA which contains –NH2 groups (Fig. 5).

Fig. 5
figure 5

Schematic illustration of carboxylic acid (–COOH) groups linker

Full size image

For example, Chen et al. (2011) reported the conjugation of dimercaptosuccinic acid (DMSA)-modified UCNP with folic acid-chitosan for targeted lung cancer imaging. The oleate-coated UCNPs were prepared according to solvothermal method just mentioned above. Surface modification of the as-synthesized NaYF4: Yb/Er nanoparticle was carried out through ligand exchange with DMSA to afford the carboxyl group. The obtained DMSA-modified UCNP was separated by centrifugation and was finally redispersed in deionized water. The DMSA-modified UCNPs with folic acid-chitosan were demonstrated to be low cytotoxicity. Further biological evaluation showed a good targeted performance in H460 lung cancer imaging (Fig. 6).

Fig. 6
figure 6

Schematic illustration of modified nanoparticle

Full size image

Another research group developed a multifunctional nanoparticle-probe for imaging. The PEGylated PLGA-modified magnetic nanoparticles, conjugated with therapeutic drugs such as herceptin or Dox, have demonstrated ultrasensitive targeted performance and excellent synergistic effects in vitro and in vivo (Yang et al. 2007) (Fig. 7). Kumar and Zhang (2009) reported a new design of DNA biosensor by using upconversion nanoparticle (NaYF4: Yb3+, Tm3+) as donor and dye (SYBR Green I) as acceptor. The test principle of detection was considered to be luminescence resonance energy transfer. The DTPA-modified UCNP exhibited good hydrophilic property and can attach to single-stranded DNA (ssDNA) probe perfectly with the help of EDC. Then DTPA-UCNP-ssDNA probe and SYBR Green I were both present in the same solution. The quantum yield of SYBR Green I was rather low unless DTPA-UCNP-ssDNA probe met target DNA. Later, Liu et al. (2011a) modified UCNP with carboxylic acid groups by using poly (acrylic acid) (PAA) for further conjugation with ssDNA. Such amino groups on ssDNA were attached to the UCNP surface successfully with EDC/NHS-assisted standard procedures.

Fig. 7
figure 7

Scheme illustration of modified nanoparticles

Full size image

Amino (–NH2) groups linker used in nanoparticle-based probe

As an important functional group in bioconjugation, –NH2 group is present in many ligands coated on the surface of nanoparticle, such as PEI, diamino PEG, PAMAM, etc. Inspiringly, the –NH2 group is particularly suitable for conjugation with target agent containing –COOH groups such as antibodies, folic acid, peptides, DNA, etc., (Fig. 8).

Fig. 8
figure 8

Schematic illustration of Amine (–NH2) groups linker

Full size image

Generally, nanoparticles (NP) with silane shell can be easily amino-modified on the surface, which is facilitated by silane coupling agent, like 3-aminopropyltrimethoxysilane (APTES). Lu et al. (2004) reported a method of preparing streptavidin-nanoparticle probe with optical and magnetic properties. The nanoparticle composites were firstly synthesized and modified with silane like TEOS. Subsequently, the nanoparticle composites were covalently coupled with streptavidin via an APS-glutaraldehyde linker. A series of experiments have confirmed that streptavidin was conjugated successfully with the nanoparticle composites. In 2005, Veiseh et al. (2005) introduced a dual-modality nanoparticle-probe for targeting gliomas. APTES–PEG–NH2 was used to provide amine group. The nanoparticle-probe was synthesized by covalently binding SPIO with APTES–PEG–NH2, which was subsequently conjugated with chlorotoxin (CTX) and the NIR dye Cy5.5. The dual-modality nanoparticle-probe showed preferential targeting abilities and high stability in gliomas imaging (Fig. 9).

Fig. 9
figure 9

Schematic illustration of modified SPIO

Full size image

Wang and Li (2006) adopted a novel green upconversion nanoparticle for DNA detection. As is shown in Fig. 10, both magnetic particle and upconversion nanoparticle were amino-modified by polyelectrolyte using layer-by-layer technology (Hong et al. 2004). The conjugation of the modified nanoparticle with nucleic acids was achieved according to the reported method (Abe et al. 2003). The modified UCNP showed many excellent properties such as low background, photostability, and chemical stability. These excellent properties of probe together hold the promise of observing molecular interactions and transportation in living cells.

Fig. 10
figure 10

Scheme illustration of the preparation of the DNA detector

Full size image

Chatterjee et al. (2008) reported the polyethylene-amine(PEI)-modified UCNP (NaYF4: Yb3+, Er3+) in vitro imaging of cancer cells and in vivo imaging in tissues. In order to equip nanoparticle with targeted performance, folic acid was conjugated to the PEI-modified UCNP (NaYF4: Yb3+, Er3+). The imaging experiment in vitro and in vivo clearly showed that modified UCNP was stable, nontoxic, and resistant to photobleaching and of good targeting property. This is the first time that UCNP was applied as a signal agent in small animal imaging. Yu et al. (2010) reported a preparation of CTX: UCNP probe using a PEI linker. As is shown in Fig. 11, the PEI-modified UCNP with amino groups on the surface and CTX peptide with carboxyl groups were cross-linked using a standard protocol. After the conjugation between UCNP and CTX, the fluorescence properties of UCNP does not change too much, and the resulting CTX: UCNP nanoparticle probe was stable for several days when dispersed in aqueous solution.

Fig. 11
figure 11

Schematic illustration of the preparation of CTX: UCNP nanoparticle-probe

Full size image

The amino-modified nanoparticle can also react with agents containing –CHO or S=C=N groups for further conjugation. Bogdan et al. (2010) used PAMAM-modified UCNP for lectin recognition. PAMAM served as an amino linker in nanoparticle conjugation. PAMAM-UCNP was synthesized by a ligand-exchange reaction between UCNP (NaGdF4:Er3+, Yb3+) and PAMAM. And at the last of their article, they introduced that the UCNP was successfully modified with both hydrophilic PAMAM and mannose to get hydrophilic nanoparticle with high biocompatibility and excellent optical properties (Fig. 12).

Fig. 12
figure 12

Schematic illustration of preparation of PAMAM–UCNP

Full size image

Maleimide (MA) groups linker used in nanoparticle-based probe

It is well known that thiol is a common group in many biomolecules such as peptides. So linker with MA groups, which can react quantitatively with thiol during the conjugation, is quite influential (Fig. 13).

Fig. 13
figure 13

Schematic illustration of maleimide (MA) groups linker

Full size image

Amine-modified nanoparticle usually can be transformed into maleimide-modified ones by reacting with a bifunctional linker. Xiong et al. (2009a) reported a bifunctional linker to convert amine-modified UCNP to maleimide-modified equivalents. Subsequently, the c(RGDFK) with thiol was conjugated with UCNP for targeted imaging through maleimide groups’ linker. By the use of the EDC/NHS-assisted standard procedure, PEG was attached to the surface of UCNP. UCNP–PEG–NH2 was further conjugated to a bifunctional linker, 6-maleimidohexanoic acid N-hydroxysuccinimide ester. Thiolated c (RGDFK) was attached to maleimide-modified nanoparticle to afford RGD-conjugated UCNP. The RGD-conjugated UCNP could be well dispersed in both hydrophilic solvents and hydrophobic solvent due to the presence of linker. The imaging experiments in vitro and in vivo showed that maleimide-modified nanoparticle had a nice coupling activity with the target agent (Fig. 14).

Fig. 14
figure 14

Schematic illustration of the preparation of RGD–UCNP

Full size image

Ryu et al. (2010) reported that the conjugation of Ni-nitrilotriacetate (NiNTA) and UCNP (NaGdF4: Yb3+, Er3+, Tm3+) was achieved by a bifunctional linker, sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC). It was found that both the hydrophobic and hydrophilic UCNP showed excellent upconverting and magnetic properties in the optical and MRI. These results indicated that the surface modification of UCNP was successful. Additionally, sulfo-SMCC has also showed its potential as a surface modification linker for the design of nanoparticle-based probe (Fig. 15).

Fig. 15
figure 15

Schematic illustration of other groups linker

Full size image

Other groups linker used in nanoparticle-based probe

In 2009, Huang et al. (2009) reported a one-step method to conjugate a lung cancer-targeting peptide with SPIO. In order to connect the SPIO surface with the targeting-peptide, a PEG linker with thiol was attached to the peptide. In this way, a cysteine residue was disposed at the C-terminus of the peptide. And then, the modified peptide was able to attach to the as-synthesized SPIO through direct ligand exchange. The Prussian blue staining and MRI both confirmed that nanoparticle-probe targeted the αvβ3 over expressed in human H2009 cancer cells nicely (Fig. 16).

Fig. 16
figure 16

Schematic illustration of modified SPIO

Full size image

In addition to the linkers noted above, there are also some other groups linkers used in nanoparticle-based probe (Veiseh et al. 2010; Erathodiyil and Ying 2011) (Fig. 15). Hayashi et al. (2009) innovatively introduced “click-chemistry” as a universal method into nanoparticle surface modification. With this method applied, azides can be developed as another type of linker for the conjugation of nanoparticle with target agents. A case in point, Fe3O4 nanoparticle was converted to azide-modified Fe3O4 nanoparticle through a series of surface modifications (Hayashi et al. 2010; Liu et al. 2011a, b). Finally, alkyne-bound folic acid was clicked onto the Fe3O4 nanoparticle surface with a high yield.

Biological characteristics of nanoparticle-based probe

As potential imaging agents, the toxicity of modified nanoparticles has been investigated with reference to in vitro cytotoxic activity and long-term living toxicity. Numerous studies have already suggested that modified nanoparticles have a low immediate toxicity when used within a certain range of concentrations and within a limited incubation period (Gupta and Curtis 2004; Nam et al. 2011; Chen et al. 2011; Zhou et al. 2012). The solubility and biocompatibility of modified nanoparticles in biological media also affect the efficiency of nanoparticles in the applications of molecular imaging. Numerous studies have been done to evaluate the changes of solubility and biocompatibility before and after the surface modification. To be sure, with the help of proper modifications, the solubility and biocompatibility have been markedly improved (Gupta and Curtis 2004; Xiong et al. 2009a; Reddy et al. 2012). However, few evaluations have been discussed about the details of solubility and biocompatibility in biologically environment by different modifications. Therefore, further understanding of the surface modification is needed.

To be sure, with the help of proper modifications, the solubility and biocompatibility have been markedly improved. Numerous studies have been done to evaluate the changes of solubility and biocompatibility before and after the surface modification. The details can be found in the following references (Gupta and Curtis 2004; Nyk et al. 2008; Kobayashi et al. 2009; Xiong et al. 2009a; Wang et al. 2010; Reddy et al. 2012; Zhou et al. 2012).

Conclusion and perspective

Over the past decades, nanoparticle-based probes have attracted a great deal of interest. As signal agent, many kinds of nanoparticles like ferric oxide particles have been extensively studied in vitro and in vivo for several years. And the surface modifications of nanoparticle have completely changed molecular imaging field. It makes that carrying-out nanoparticles with unique properties in bioimaging become possible. In this review, the surface modification of how nanoparticle assembles to molecular imaging probe has been discussed. Particularly and intensively, two kinds of nanoparticle used in wide range within molecular imaging field were discussed in this paper. On the other hand, chemistry has demonstrated its power in preparing nanoparticle-based probes with controlled size, shape, morphology, and surface modifications. However, surface modifications sometimes could compromise some advantages of nanoparticle. For example, after the surface modification transfer nanoparticle from hydrophobic to hydrophilic, the magnetic/optical signal would probably decrease, or the potential long-term toxicity may change. That means much more time is needed devoting to further research.

Taken together, the design of molecular imaging probe has always been an exciting field filled with both challenges and opportunities. Medical research from bench to bed has been tremendously influenced by the efforts and achievements made in this scope. Moreover, with the development of nanotechnology, continuous discoveries of new types of nanoparticle or new technology, coupled with their application in surface modification, will undoubtedly help to improve the clinical treatment. As for the nanoparticle-based probe design, though lots of accomplishments have been made over the past decades, it is clear that there are still many problems remained to be solved. The safety, biocompatibility, stability, and targeting performance in vitro and in vivo all need to be addressed by a suitable surface modification approach. We expect that such challenges could help us a better understanding of nanoparticle application in molecular imaging.